Abstract One important yet understudied aspect in nanomaterial applications is the cell biology of nanomaterial transport into cells and through tissues. To overcome the cell membranes and reach their sites of action within, nanoparticles (NPs) are often complexed with cell-penetrating ligands. Such ligands include peptides like TAT (Trans-Activating Transcription). These ligands engage with cell surface receptors to invoke endocytic processes for NP uptake. However, the impact of these functionalized NPs and the resulting endocytic process on the cellular uptake of other NPs remains unknown. In this proposal, I aim to tackle this problem by studying TAT-coupled NPs (TAT-NP). By tracing NPs coated with several cell-penetrating peptides, including TAT, we previously discovered a novel receptor-dependent macropinocytosis (MP) pathway for NP uptake. MP has relatively large endocytic vacuoles (>200 nm in diameter) and thus can more readily engulf cargo as large as nanoparticles. Here, we found that TAT-NPs, by invoke this MP pathway, can bring into cells bystander NPs that are unable to enter the cells by themselves. This bystander uptake depends on TAT interaction with its receptor, heparan sulfate (HS) proteoglycans, and occurs through the HS-dependent MP pathway. We further showed that it is only active for NP-type bystander cargo, and its activity is greatly stimulated by Cysteine (Cys) outside the cells. Here, we aim to dissect out this bystander uptake process. In the Aim 1, we will determine the prerequisites for initiating the bystander uptake. In the Aim 2 and 3, we will utilize genetic screening and other methods to determine the factors that mediate this bystander uptake, and define its properties of cargo selectivity and Cys regulation. In the Aim 4, we will explore the potential use in biomedical applications. My proposed studies hold great potential for unveiling important cellular machineries for nanomaterial transport, boosting the efficiency of intracellular delivery, and opening up a new avenue to study cell biology, metabolism and nanomaterial delivery.